Eucommia ulmoides and Tribulus terrestris (ET) has been widely utilized in clinical practice for thousands of years, but the mechanism underlying its efficacy has not been elucidated to date. This study attempted to investigate the role played by the intestinal microbiota and fecal metabolism in the response of elderly spontaneous hypertensive rats (SHRs) to ET administration as a treatment for hypertension. Fourteen male spontaneously hypertensive rats  were randomly divided into an ET group and an SHR group, and 7 Wistar-Kyoto (WKY) rats of the same age were employed as the control group. The ET group was intragastrically administered 1.0 g/kg/d ET for 42 days, and SHRs and WKY rats were administered an equal amount of normal saline intragastrically. The intestinal microbiota and fecal metabolism were analyzed by 16S rRNA sequencing and the GC-MS (gas chromatography-mass spectrometry)/MS assay. ET treatment decreased blood pressure steadily, improved the colonic tissue morphology, and changed the structure and composition of the imbalanced microbiota in SHRs. Specifically, ET treatment increased the abundance of Eubacterium, which might be one of the target microbes for ET, and had a negative correlation with the levels of α-tocopherol, chenodeoxycholic acid, and deoxycholic acid according to the Spearman correlation analysis. The change in the intestinal microbiota affected the fecal metabolic pattern of SHRs. Eight potential biomarkers were determined to be primarily enriched in ABC transporters, phenylalanine metabolism, central carbon metabolism in cancer, purine metabolism, and protein digestion and absorption. The correlation analysis demonstrated that the abundance of Eubacterium and the decreased levels of α-tocopherol, chenodeoxycholic acid, and deoxycholic acid in the ET group were highly correlated. Our results suggest that ET has a good antihypertensive effect, which may be driven by the intestinal microbiota and their beneficial metabolites. The results of this study may help to elucidate the antihypertensive mechanism of ET.The combination of  Accumulating evidence indicates that there is a close relationship between the intestinal microbiota and hypertension. The intestinal microbiome, which is considered the second human genome , 2, is bEucommia ulmoides, the bark of Eucommia ulmoides Oliv., has the Chinese name Duzhong and the English name Eucommiae Cortex. The use of this bark has been recorded for thousands of years, and it is used to improve general health in traditional Chinese medicine. Pharmacological studies showed that aqueous and alcohol-based extracts of Eucommia ulmoides had the actions of lowering blood pressure and promoting bone-healing, antiaging, and vasodilatory effects. Tribulus terrestris has the Chinese name Cijili and the English name Puncturevine Caltrop Fruit. This plant grows widely in subtropical regions and is generally utilized to treat hypertension, arteriosclerosis, and aging. Our previous studies indicated that Tribulus terrestris decreases the generation of reactive oxygen species while increasing the concentration of NOS and improving the metabolism of endothelial cells [Eucommia ulmoides and Tribulus terrestris (ET) has been widely employed in clinical practice in traditional Chinese medicine. As one of the combination drugs for the treatment of hypertension (patent number: CN201810790515.4), ET exhibited good efficacy against age-related hypertension in previous studies [al cells . The com studies , 14.In this study, we investigated the effects of ET on the intestinal microbiota and its metabolites. We sequenced the 16S rRNA V4 region in the intestinal microbiota and performed gas chromatography-mass spectrometry (GC-MS) on fecal metabolites and integratively analyzed their correlation to explore the potential mechanism underlying the antihypertensive effects of ET.Eucommia ulmoides and Tribulus terrestris  were purchased from the Affiliated Hospital of Shandong University of Traditional Chinese Medicine. The formula particles were dissolved with ultrapure water to a concentration of 1.63 g raw herbs/ml .Formula particles of No. AF/SC-08/02.0), Affiliated Hospital of Shandong University of Traditional Chinese Medicine . All efforts were made to alleviate the pain of experimental animals. All animals and experimental procedures in this study were performed in keeping with the Regulations on the Management of Experimental Animals of People's Republic of China . Fourteen male spontaneously hypertensive rats  and 7 WKY rats of the same age were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (certificate: SCXK (Jing) 2016-0011). The rats were maintained under the conditions of constant temperature (20-22°C), constant humidity (50-60%), and fixed light and dark alternation (12 h/12 h). All rats had access to Cobalt 60-sterilized fodder  and ultrapure water ad libitum. After a 5-day adaptive feeding, SHRs were randomly divided into the ET group and the SHR group. SHRs in the ET group were given 1.0 g/kg/d ET suspension  intragastrically, which was equivalent to 0.5 g/kg/d Eucommia ulmoides + 0.5 g/kg/d Tribulus terrestris in human clinical application, for 6 weeks. The WKY rats and SHRs were administered the same volume of normal saline.The study was approved by the Faculty of Medicine and Health Sciences Ethics Committee for Animal Research . Rats were allowed to adapt to the pressure measurement environment for 1 week before formal recording. Rats were placed in the ALC-HTP animal system in quiet and awake states. The systolic pressure and heart rate of the rats were measured. The average of 5 measurements was recorded in parallel with an interval of 1 min. Stool samples were collected by pinching the tail to let rats defecate under stress and collected from the anus to a sterile centrifuge tube directly. All samples were stored at -80°C.μm, stained with hematoxylin-eosin (HE), and sealed with neutral balsam. The slides were observed and photographed under a light microscope.The colons were removed on ice and fixed in neutral paraformaldehyde for 48 h. The colon tissues were embedded in paraffin, sectioned at a thickness of 4 Sequencing of 16S rRNA was performed by Novogene Technology Co., Ltd. .μl. The V4 region within the 16S rRNA gene was amplified using specific primers. The primer sequences were 5′-GTGCCAGCMGCCGCGGTAA-3′/5′-GGACTACHVGGGTWTCTAAT-3′. PCR products were confirmed with 2% agarose gel electrophoretic analysis, and samples with bright and distinct bands between 400 and 450 bp were extracted and purified with a Qiagen Gel Extraction Kit .DNA concentration and purity were determined on 1% agarose gels. The DNA concentration was adjusted to 1 ng/Sequencing libraries were generated using the TruSeq® DNA PCR-Free Sample Preparation Kit  following the manufacturer's recommendations. The library quality was assessed on the Qubit@ 2.0 Fluorometer  and Agilent Bioanalyzer 2100 system. Finally, the library was sequenced on an Illumina HiSeq 2500 platform, and 250 bp paired-end reads were generated.Paired-end reads were assigned to samples based on their unique barcode and were truncated by cutting off the barcode and primer sequence. For sequence assembly, paired-end reads were merged using FLASH (V1.2.7). High-quality clean tags were utilized according to the QIIME (V1.7.0) quality-controlled process. The tags were compared with the reference database (Gold database) using the UCHIME algorithm (UCHIME Algorithm) to detect and remove chimeric sequences. Next, the effective tags were obtained. Sequence analysis was performed by Uparse software (Uparse v7.0.1001). Sequences with ≥97% similarity were assigned to the same OTUs. For each representative sequence, Green Gene Database 3 was used based on the RDP classifier (version 2.2) algorithm to annotate taxonomic information. Multiple sequence alignment was performed using MUSCLE software (version 3.8.31).Alpha diversity was analyzed by PD-whole tree and observed species. Beta diversity was manifested as PCoA and NMDS analysis. LEfSe analysis was employed to compare species with significant differences among groups. The Spearman correlation coefficient was calculated to determine the potential correlation between the intestinal microbiota and metabolites.The GC-MS assay was performed by OE Biotech Co., Ltd. .μm, Agilent J&W Scientific, Folsom, CA, USA) was utilized to separate the derivatives at a constant flow of 1 ml/min helium. One microliter of sample was injected in split mode at a 20 : 1 split ratio by the autosampler. The injection temperature was 280°C, the interface was set to 150°C, and the ion source was adjusted to 230°C. The temperature rise program was followed by an initial temperature of 60°C for 2 min, a 10°C/min rate up to 300°C, and a temperature of 300°C for 5 min.A DB-5MS fused-silica capillary column (30 m × 0.25 mm × 0.25 m/z).Mass spectrometry was performed using the full-scan method with a range of 35 to 750 . Partial least square analysis (PLS-DA) and orthogonal partial least square analysis (OPLS-DA) were utilized to distinguish the overall differences in metabolic profiles among groups and to identify the different metabolites. Student's p values less than 0.05 were considered to be significantly different. The Pearson correlation coefficient was utilized in the correlation analysis between the intestinal microbiota and fecal metabolites.Statistical analyses were performed using SPSS 20.0. The values are presented as the means ± standard deviation. Unless otherwise stated, statistical comparisons were performed using a single-factor analysis of variance (ANOVA) followed by the LSD test. In this study, p > 0.05). After 6 weeks of treatment, both systolic and diastolic blood pressures in the ET group were significantly lower than those in the SHR group (p < 0.05)  . As showα-diversity and β-diversity to describe the intra- and intergroup differences of microbiota. The results of PD-whole tree and observed species analysis (α-diversity) showed that both the abundance and diversity of the intestinal microbiota among the three groups could be ranked as follows: WKY > ET > SHRs  and a nonlinear model . Both PCoA analysis and NMDS analysis showed that there were different clusters among the three groups, suggesting significant community differences -3,4-dihydroxybutyric acid, 2-hydroxybutyric acid, 6-deoxyglucose, chenodeoxycholic acid, deoxycholic acid, D-fructose, and D-fructose-6-phosphate and a negative correlation with 1-monostearin, 2-desoxy-pentos-3-ulose, 2-piperideinobenzonitrile, and 4′,5-dihydroxy-7-glucosyloxyflavanone.Erysipelotrichaceae had a positive correlation with the metabolic level of 1-monostearin and 4′,5-dihydroxy-7-glucosyloxyflavanone and a negative correlation with 1-hexacosanol, 3,6-anhydro-D-hexose, 5-alpha-cholestanol, alpha-tocopherol, chenodeoxycholic acid, deoxycholic acid, and D-fructose-1-phosphate and D-fructose-6-phosphate.Unidentified_Lachnospiraceae had a positive correlation with the metabolic level of 1-monostearin and 2-piperidinobenzonitrile and a negative correlation with 1-hexacosanol, 2-hydroxybutyric acid, 3,6-anhydro-D-hexose, 4-hydroxybenzeneacetic acid, 5-alpha-cholestanol, chenodeoxycholic acid, deoxycholic acid, D-fructose, D-fructose-1-phosphate, and D-fructose-6-phosphate  [Lactobacillus is a microorganism to which antihypertensive effects have been attributed [Streptococcus and Lactobacillus were significantly increased after ET administration, which supports the efficacy of ET in lowering blood pressure.rtension , while bcontrol) . Lactobatributed . In the α-tocopherol at a moderate level has been indicated to have an anti-inflammatory effect. However, treatment with excessive quantities of α-tocopherol increased the risk of mortality in humans according to a meta-analysis [+/K+) ATPase in a dose-dependent manner, and the altered Na+/K+ ratio is related to hypertension [α-dehydroxylated enzyme and 7β-dehydroxylated enzyme, which both participate in the process of bile acid conversion to deoxycholic acid and other secondary metabolites, are completely involved in the intestinal microbiota [α-tocopherol, chenodeoxycholic acid, and kynurenine were downregulated after ET administration, while enterolactone was upregulated. Other metabolites, such as deoxycholic acid, fumaric acid, N-acetylgalactosamine, and nonanoic acid, were also downregulated. The mechanism governing the involvement of these metabolites in blood pressure regulation has not been elucidated. Remarkably, changes in those metabolites were at least partly regulated by the intestinal microbiota [Treatment with analysis . A studyrtension . Chenodertension . The actcrobiota . Notablycrobiota . Kynurencrobiota . Enterolcrobiota . Interescrobiota . In the crobiota , 29. It crobiota .Our fecal metabonomics data suggested that metabolites affected by ET were primarily enriched in ABC transporters, phenylalanine metabolism, central carbon metabolism in cancer, purine metabolism, L-tyrosine digestion and absorption, mineral absorption, taste transduction, renal cell carcinoma, fatty acid biosynthesis, and aminoacyl-tRNA biosynthesis.ABC transporters contribute primarily to energy metabolism and transport various substrates, including ions, peptides, lipids, and proteins, involving many key physiological and pathological processes. Although these transporters have not been related to hypertension, they are closely related to atherosclerosis, gout and hyperuricemia, coronary heart disease, and other diseases that are high-risk factors for hypertension , 30, 31.It is worth noting that phenylalanine metabolism is a precursor to catecholamine biosynthesis. There is an abnormal state of phenylalanine metabolism in spontaneously hypertensive rats. The intake of aromatic amino acids, including phenylalanine (AAAs), increases the risk of hypertension according to a clinical study of 4288 individuals . In our Abnormal purine metabolism is an independent risk factor for hypertension. Hyperuricemia is caused by abnormal purine metabolism, which leads to a decrease in nitric oxide synthesis, changes in the biological behavior of vascular smooth muscle cells, inflammation, oxidative stress, and an increased risk of hypertension. ET treatment effectively downregulated purine metabolism levels.Fatty acid metabolism has multiple effects on the development of hypertension. Short-chain fatty acids are among the primary products of intestinal microbial metabolism, and they affect the blood pressure level of the host through Gpr41. Acute short-chain fatty-acid administration can decrease the blood pressure levels of spontaneously hypertensive rats , improveEucommia ulmoides and Tribulus terrestris are considered to be kidney tonifying agents in traditional Chinese medicine.There is a definite relationship between the pathogenesis of hypertension and cancer. These diseases share similar risk factors and pathophysiology mechanisms. Hypertension increased the risk of mortality from cancer, particularly renal cell carcinoma, according to a retrospective study conducted in 2002 . In the Minerals are necessary elements to maintain physiological activities. It is known that the levels of certain elements, such as Ni and Co, in the serum are negatively correlated with systolic and diastolic blood pressure, while the serum Se level is positively correlated with blood pressure. Physical function declines with aging, and disorders of mineral absorption and metabolism are exacerbated, which is not conducive to the regulation of blood pressure. In this study, the body's absorption of minerals was significantly improved after ET administration, which was a notable example of the multitarget effect of traditional Chinese medicine.Taste transduction is related to neuroendocrine regulation, and the mechanisms of sensing taste in the oral cavity have been thoroughly characterized. Several studies  have demα-tocopherol, chenodeoxycholic acid, and deoxycholic acid were highly closely correlated with the abundance of Eubacterium according to the Spearman correlation analysis. A clinical study also showed that the number of Eubacterium rectale bacteria was increased in the intestines of patients with hypertension [Eubacterium after the administration of ET. This microbe may be one of the target microbiota of ET that play important roles in lowering blood pressure. The effects of Erysipelotrichaceae on the host still warrant further research; however, considerable evidence [Erysipelotrichaceae is related to lipid metabolism, which is consistent with our results. In addition, Erysipelotrichaceae in SHRs also exhibited a correlation with flavonoid metabolism. It is known that flavonoids are important pharmacophore monomers in both Eucommia ulmoides and Tribulus terrestris [Erysipelotrichaceae was identified as a biomarker of ET in this study, we speculated that ET may regulate blood pressure by targeting lipid and flavonoid metabolism by increasing the abundance of Erysipelotrichaceae. Meanwhile, an increase in the abundance of Erysipelotrichaceae after ET treatment helped to regulate the structure of the intestinal microbiota of SHRs. We determined that ET treatment affected metabolism and enhanced the utilization of flavonoids in SHRs.The correlations between rtension . Our stuevidence  indicaterrestris . Flavonorrestris . BecauseEucommia ulmoides and Tribulus terrestris yielded a good antihypertensive effect, and this efficacy was related to the improvement of the intestinal microbiota and their related metabolites. We hope that these results serve to elucidate the antihypertensive effects of the combination of Eucommia ulmoides and Tribulus terrestris.The combination of


Brucella spp. have been associated with abortion in humans and animals. Although the mechanisms involved are not well established, it is known that placental Brucella infection is accompanied by inflammatory phenomena. The ability of Brucella abortus to infect and survive in human endometrial stromal cells (T-HESC cell line) and the cytokine response elicited were evaluated. B. abortus was able to infect and proliferate in both non-decidualized and decidualized T-HESC cells. Intracellular proliferation depended on the expression of a functional virB operon in the pathogen. B. abortus internalization was inhibited by cytochalasin D and to a lower extent by colchicine, but was not affected by monodansylcadaverine. The infection did not induce cytotoxicity and did not alter the decidualization status of cells. B. abortus infection elicited the secretion of IL-8 and MCP-1 in either decidualized or non-decidualized T-HESC, a response also induced by heat-killed B. abortus and outer membrane vesicles derived from this bacterium. The stimulation of T-HESC with conditioned media from Brucella-infected macrophages induced the production of IL-6, MCP-1 and IL-8 in a dose-dependent manner, and this effect was shown to depend on IL-1β and TNF-α. The proinflammatory responses of T-HESC to B. abortus and to factors produced by infected macrophages may contribute to the gestational complications of brucellosis. Brucella melitensis, B. suis and B. abortus, affects over 500,000 people each year around the world [Human brucellosis, a zoonotic disease mostly caused by he world . The infBrucella infection is accompanied by the infiltration of inflammatory cells [Brucella-induced abortion.The clinical manifestations of human brucellosis are usually linked to inflammatory phenomena in the affected organs . Involvery cells ,4. The fry cells ,6,7 suggBrucella infection has been also reported in humans, with an incidence that ranges from 7% to 40% according to different studies [Brucella-related abortion, the pathophysiology of this complication in humans is largely unknown. Recent studies have shown that Brucella spp. can infect and replicate in human trophoblasts, and that the infection elicits a proinflammatory response [Brucella infection has not been explored. Abortion due to  studies ,9,10. Am studies . In spitresponse ,13. ThesBrucella abortus, in vivo studies in animal models have indicated that the maternal decidua is the initial site of placental colonization [For several microorganisms that reach the placenta by the hematogenous route, including nization ,15. Decinization .Given the relevance of the decidua as the initial site of placental colonization for several hematogenously spread infections, the ability of decidual cells to respond to pathogens is especially relevant. Primary DSC and ESC cell lines have been shown to express several Toll-like receptors (TLRs) and Nod-like receptors (NLRs), and respond to pathogen-associated molecular patterns (PAMPs) with an enhanced production of matrix metalloproteinases (MMPs) and proinflammatory cytokines including MCP-1, IL-6, IL-8, IL-1β, and CCL5 (RANTES) . At leasEscherichia coli, a coculture of ESC and PMA-differentiated THP-1 cells (human monocytes) produced enhanced levels of many cytokines  as compared to the respective monocultures [B. abortus is known to survive and replicate in macrophages from several animal species, inducing the secretion of proinflammatory cytokines [In addition to decidual stromal cells, the decidua also contains significant proportions of immune cells, including macrophages, natural killer cells, dendritic cells, and T cells . Early pcultures . Importaytokines ,26,27. Brucella spp. to infect and survive in decidualized T-HESC, and also assessed the cytokine production induced in these cells by the infection or by their interaction with infected macrophages.The T-HESC cell line, derived from normal primary human ESC by telomerase immortalization, has been widely used to study several aspects of human ESC biology, including infection and cytokine production ,29,30,31B. abortus at a multiplicity of infection (MOI) of 250 bacteria/cell, and colony-forming units (CFU) of intracellular bacteria were determined at different times post-infection (p.i.). As shown in B. abortus was able to infect T-HESC cells in both conditions, although the initial number of intracellular bacteria (2 h p.i.) was slightly higher for non-decidualized cells . Besides wild type B. abortus, two additional strains carrying mutations in genes relevant for virulence were also tested for their capacity to infect and survive in T-HESC cells. These included a mutant lacking the virB10 gene, widely reported as essential for the intracellular survival and replication of Brucella [btpA and btpB genes which encode proteins able to interfere with TLR signaling [virB10 mutant and the other two strains. While CFU of intracellular bacteria increased along time for wild type B. abortus and the btpAbtpB mutant, showing intracellular replication, the CFU of the virB10 mutant declined at the same time and no viable bacteria were detected in either condition at 48 h p.i. This later result confirmed in endometrial cells the essential role of virB10 for the intracellular survival of Brucella.Both decidualized and non-decidualized T-HESC endometrial cells were infected with Brucella ,34, and ignaling ,36. As sB. abortus internalization by T-HESC cells depends on actin polymerization , microtubules (colchicine), or clathrin-mediated endocytosis . As shown in B. abortus internalization was highly inhibited by cytochalasin D and to a lower extent by colchicine, but was not affected by MDC. Infection experiments were also carried out in the presence of specific inhibitors to examine whether B. abortus or the btpAbtpB mutant did not modify the levels of LDH or prolactin as compared to non-infected cells at any time point, showing that it does not induce cytotoxicity or affect the decidualization of cells.To assess whether the infection affected the viability of T-HESC cells or their decidualization status, the levels of lactate dehydrogenase (LDH) and prolactin were measured in culture supernatants of infected cells at 24 and 48 h p.i. and also in non-infected cells cultured in parallel. As shown in B. abortus to induce a proinflammatory response in T-HESC, these cells were infected with the wild type strain and the btpAbtpB mutant, and the levels of IL-8 and MCP-1 were measured in culture supernatants. The studies were performed on decidualized and non-decidualized cells to determine whether the proinflammatory response depends on the decidualization status. As shown in B. abortus strains elicited the secretion of both chemokines in either decidualized or non-decidualized T-HESC, and this effect was mostly evident at 48 h p.i. At this time point, IL-8 levels were higher in non-decidualized cells than in decidualized ones , whereas no significant difference was found for MCP-1 .As mentioned above, DSC and ESC cell lines express several TLRs and NLRs, and respond to microbial PAMPs with an enhanced production of proinflammatory cytokines, including MCP-1, IL-6, IL-8, IL-1β, and RANTES . To asseB. abortus, and IL-8 and MCP-1 were measured as above. As shown in To determine which signaling pathways may be involved in the induction of chemokine secretion, decidualized T-HESC cells were treated with SB203580 (p38 MAPK inhibitor), SP600125 (Jnk1/2 inhibitor), BAY 11-7082 (NF-κB inhibitor), or vehicle  before and during the infection with B. abortus infection to induce the secretion of IL-8 and MCP-1 in T-HESC cells, experiments were carried out to determine whether such responses can be also elicited by stimulation with B. abortus antigens or, conversely, depend on bacterial viability. For this purpose, cells were stimulated with either heat-killed B. abortus (HKBA), or LPS or outer membrane vesicles (OMVs) from this bacterium, and chemokine levels were measured at 48 h p.i. As shown in 9 CFU/mL) elicited IL-8 and MCP-1 secretion by T-HESC cells, albeit at lower levels than those attained by the infection. In addition, IL-8 secretion was significantly induced by B. abortus OMVs. These results show that the induction of chemokines in these cells does not depend on Brucella viability.Given the ability of B. abortus or stimulation with its antigens. In the context of infection in the pregnant uterus, however, endometrial cells may also receive stimulation by factors produced by adjacent infected macrophages [B. abortus-infected macrophages and the levels of proinflammatory cytokines were measured in culture supernatants 24 h later. The preexisting levels of these cytokines in the CM were subtracted in order to calculate the secretion specifically induced by the stimulation. As shown in Brucella-infected macrophages induced the production of IL-6, MCP-1, and IL-8 in a dose-dependent manner (higher secretion for stimulation with CM diluted at 1/2). No significant secretion of any of these cytokines was induced by stimulation with CM from non-infected monocytes. Previous similar studies on the stimulation of other non-phagocytic cells have shown that IL-1β and TNF-α are involved in the inducing effect of CM from Brucella-infected macrophages. To test whether these cytokines are also involved in the stimulation of IL-6, IL-8, and MCP-1 in decidualized T-HESC cells, experiments were performed in which CM were preincubated with a TNF-neutralizing antibody or T-HESC were preincubated with the natural antagonist of the IL-1 receptor (IL-1Ra). As shown in Brucella-infected macrophages to stimulate the production of proinflammatory cytokines by decidualized T-HESC.The results shown above demonstrate that decidualized T-HESC produce proinflammatory mediators in response to infection with rophages ,23. To mBrucella infections have been associated with abortion in both humans and animals. Although the pathophysiology of this complication has not been completely elucidated, the inflammatory phenomena observed in the affected placenta [Brucella-induced abortion. As Brucella can reach the placenta by the hematogenous route, the maternal decidua is probably the initial site of placental colonization [Brucella spp. to colonize decidualized stromal endometrial cells (T-HESC) and to induce the production of proinflammatory cytokines. placenta ,4 suggesnization ,15. GiveB. abortus was able to infect both decidualized and non-decidualized T-HESC cells, although the initial number of intracellular bacteria was slightly higher for non-decidualized cells. This may relate to the fact that decidualized cells form an organized layer thus exposing less membrane surface to the environment. In addition, the pathogen was able to survive and replicate inside these cells. These findings are in line with the reported ability of B. abortus for intracellular replication in several phagocytic and non-phagocytic cells, including macrophages, epithelial cells and trophoblasts [virB operon, which allows Brucella to modulate phagosome-lysosome fusion [B. abortus mutant lacking the virB10 gene was unable to survive and replicate inside decidualized and non-decidualized T-HESC despite a similar ability of invasion compared to the wild type strain. In contrast, a mutant lacking the genes for the BtpA and BtpB proteins that interfere with TLR signaling exhibited invasion and replication abilities similar to the wild type strain. Importantly, B. abortus infection did not induce cytotoxicity, nor did it affect the decidualization status of cells, suggesting that the decidua might sustain the infection in affected individuals.As shown here, hoblasts ,25,37. Ie fusion ,34. In lBrucella invasion of non-phagocytic cells may vary according to the cell type considered. Whereas actin polymerization and microtubules have been involved in many cells [B. abortus internalization was inhibited by cytochalasin D and to a lower extent by colchicine, which inhibit actin polymerization and microtubule formation, respectively. In contrast, internalization was not affected by MDC, an inhibitor of clathrin-mediated endocytosis.The mechanisms for ny cells , internany cells . The reqBrucella infection is accompanied by the infiltration of inflammatory cells [Brucella-induced abortion as it does in abortion triggered by other pathogens. Our results show that B. abortus infection elicits the secretion of IL-8 and MCP-1 in either decidualized or non-decidualized T-HESC cells. IL-8 levels were higher in non-decidualized cells than in decidualized ones, whereas no significant difference was found for MCP-1. The higher production of IL-8 in non-decidualized cells may relate to the higher number of intracellular bacteria found in this condition as compared to decidualized cells, a downmodulating effect of decidualization on IL-8 production [B. abortus. A few previous studies have shown that Brucella BtpA and BtpB proteins, which contain TIR motifs and can thus modulate TLR signaling, can reduce cytokine production in dendritic cells in vitro  and in lung tissues in vivo  [Brucella-infected endometrial cells was unknown. At variance with those previous studies, we did not detect significant differences in the levels of the two chemokines here evaluated (IL-8 and MCP-1) between T-HESC infected with the wild type B. abortus strain or the btpAbtpB mutant strain. These results agree with those reported for the same chemokines in Brucella-infected human trophoblasts [As mentioned previously, placental ry cells ,4, whichoduction , or both, MCP-1) ,36. Howevitro IL-, TNF-α aB. abortus. For example, CCL20 secretion by human bronchial epithelial cells depends on p38, Jnk1/2, Erk1/2, and NF-kB [The secretion of both cytokines was reduced to basal levels by all the inhibitors tested, suggesting that all the signaling pathways  are involved. In line with these findings, previous studies have shown that several signaling pathways are involved in cytokine production by different cell types in response to nd NF-kB , whereasnd NF-kB . B. abortus but also some of its antigens can elicit the production of proinflammatory cytokines [B. abortus elicit IL-8 and/or MCP-1 secretion in T-HESC cells. Obviously, these findings imply that the induction of chemokines in these cells does not depend on Brucella viability. At variance with HKBA and OMVs, B. abortus LPS did not elicit the production of the chemokines analyzed. This result is in line with previous studies in other cell types, which demonstrated that B. abortus LPS is a poor inducer of proinflammatory responses [Previous studies in several non-phagocytic cells have shown that not only live ytokines ,43,44. Iesponses ,43,44,45B. abortus or stimulation with its antigens. However, the decidua contains not only DSC but also a significant proportion of macrophages [B. abortus infection in the pregnant uterus, endometrial cells may respond not only to the stimulus of bacterial antigens but also to stimulation by factors produced by adjacent Brucella-infected macrophages. In support of this hypothesis, we found that the stimulation of decidualized T-HESC with CM from B. abortus-infected macrophages induced the production of IL-6, MCP-1, and IL-8 in a dose-dependent manner, a phenomenon not produced by stimulation with CM from non-infected monocytes. Additional studies using specific blocking agents revealed that IL-6 induction by CM is mediated by TNF-α and IL-1β, whereas the induction of MCP-1 and IL-8 is mediated by TNF-α. These findings are similar to those reported for the interaction between Brucella-infected macrophages and human trophoblasts [As shown in this study, decidualized T-HESC produce proinflammatory mediators, including MCP-1, in response to infection with rophages , with whrophages ,23. In ahoblasts .B. abortus infection and/or in response to cytokines produced by Brucella-infected placental macrophages. Reciprocal stimulations between DSC and phagocytes may amplify these phenomena. These interactions may be long-lasting due to the ability of Brucella to survive and replicate within macrophages and DSC. Altogether, these proinflammatory responses may contribute to the gestational complications of brucellosis.Overall, these results suggest a possible scenario in which DSC produce IL-6 and chemoattractants for monocytes/macrophages in response to Brucella abortus 2308 was provided by Ignacio Moriyón . The purity and the characteristics of this preparation have been published previously [LPS from eviously .4 cells/well) were treated with medroxyprogesterone acetate  and dibutyryl cAMP (0.5 mM) for 8 days, changing the culture media every 48 h. Decidualization was evaluated by morphology and by prolactin levels measured by sandwich ELISA (R&D Systems). For infection assays, cells were cultured for 24 h in antibiotic-free culture medium.A human endometrial stromal cell line (T-HESC) was kindly provided Dr. Andrea Randi . This cell line was derived from normal stromal cells obtained from an adult patient subjected to hysterectomy, and conserved the characteristics of the regular endometrial cells . The ling for 30 min. The layer containing peripheral blood mononuclear cells was carefully removed by pipetting and washed with PBS by centrifugation at 250× g for 10 min. The pellet was resuspended in RPMI 1640 medium supplemented with 1 mM glutamine and was incubated for 2 h in 24-well plates. After washing with sterile PBS to eliminate nonadherent cells, RPMI medium supplemented with 10% sera from the same donors and antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin) was added to the adherent cells. Cells were incubated at 37 °C in a 5% CO2 atmosphere for 7 days for in vitro macrophagic differentiation [Peripheral blood samples were obtained from healthy volunteers after approval by the Ethics Committee of the School of Pharmacy and Biochemistry . Written informed consent was obtained from all volunteers. Human monocytes were isolated by standard procedures. Briefly, whole blood diluted with sterile phosphate-buffered saline (PBS) was carefully layered on Ficoll-Paque (density: 1.077 g/mL) and centrifuged at 400× ntiation . AntibioB. abortus 2308 (wild type strain), its isogenic btpAbtpB double mutant and virB10 polar mutant were obtained from our collection. The strains were grown in tryptic soy broth at 37 °C with agitation. After two washes with sterile PBS, bacterial inocula were adjusted to the desired concentration in sterile PBS based on optical density readings. An aliquot of each suspension was plated on tryptic soy agar (TSA) and incubated at 37 °C to determine the actual concentration of colony-forming units (CFU) in the inocula. Cells were inoculated with B. abortus 2308 at an MOI of 200 and the plates were centrifuged for 10 min at 1200 rpm at room temperature. After 2 h, culture medium was removed and replaced with medium containing gentamicin and streptomycin. All live Brucella manipulations were performed in biosafety level 3 facilities. To prepare HKBA, bacteria were washed in sterile PBS, heat killed at 70 °C for 30 min, aliquoted, and stored at −80 °C until use. The absence of bacterial viability was checked by plating on TSA.B. abortus 2308 were obtained essentially as described previously [600 nm of 0.1 and cultured for 72 h (early stationary phase of growth). The culture was centrifuged, and the cell-free supernatant was filter-sterilized. The filtrate was centrifuged at 15,000× g for 5 h at 4 °C. The pellets containing the OMVs were resuspended in PBS, and protein concentration was measured by the bicinchoninic acid assay (Pierce). The presence of OMVs was corroborated by electron microscopy. OMVs were stored at −20 °C until use.OMVs from eviously . Briefly4 cells/well) were stimulated with LPS from B. abortus (1 µg/mL), OMVs (1 µg/mL of protein), or HKBA (109 or 108 CFU/mL). Cells were cultured at 37 °C in a 5% CO2 atmosphere, and supernatants were collected 48 h after stimulation for chemokine measurement. Decidualized T-HESC cells  and then incubated for 2 h at 37 °C in a 5% CO2 atmosphere. Non-internalized bacteria were eliminated by several washes with medium alone followed by incubation in medium supplemented with 100 µg/mL gentamicin and 50 µg/mL streptomycin. After that, cells were washed and then incubated with culture medium without antibiotics. At different times post-infection  culture supernatants were harvested for cytokine measurement, while the cells were washed with sterile PBS and lysed with 0.2% Triton X-100. Serial dilutions of the lysates were plated on TSA to enumerate intracellular CFU. In addition, the levels of prolactin were measured in culture supernatants as described above to assess the impact of infection on the decidualization status of the cells, and the levels of LDH were measured to assess cytotoxicity. Decidualized and non-decidualized T-HESC cells were infected with To analyze the effect of infection on cell integrity, the release of lactate dehydrogenase (LDH) from infected T-HESC cells was determined. LDH activity was determined using the CytotTox 96 Non-Radiactive Cytotoxicity Assay  in culture supernatants obtained at 24 and 48 h p.i. Results were expressed as the ratio between LDH levels measured in the samples (infected or non-infected cultures) and those corresponding to a 100% cell lysis (obtained by hypotonic lysis of the same number of cells).B. abortus internalization, decidualized T-HESC were pretreated for 1 h with different doses of colchicine , monodansylcadaverine  or cytochalasin D  and were later infected as described above but in the presence of these inhibitors. MDC and cytochalasin were solubilized in dimethyl sulfoxide (DMSO), and in all the experiments control cells were incubated without inhibitor or with DMSO for the same period as treated cells. Intracellular CFU were determined at 2 h p.i. as described above.To assess the role of microtubules, actin or clathrin in B. abortus 2308 (MOI 100) were harvested at 24 h p.i., filter-sterilized and used to stimulate noninfected decidualized T-HESC cells. After 24 and 48 h, supernatants from stimulated cultures were harvested to measure cytokines. The preexisting levels of cytokines in the CM were subtracted in order to calculate the secretion specifically induced by the stimulation. To determine if TNF-α might be involved in the stimulating effects of CM, in some experiments CM were preincubated for 1 h at 37 °C with a neutralizing monoclonal antibody against TNF-α or its isotype control (both from BD Pharmingen) before being transferred to T-HESC cultures. Alternatively, to determine the role of IL-1 β in the stimulating effect, decidualized T-HESC cells were preincubated with the IL-1β receptor antagonist IL-1Ra (R&D Systems) for 1 h at 37 °C before stimulation with CM from infected macrophages.CM from macrophages infected with B. abortus and were kept throughout the experiment (48 h). Cell viability after incubation with these inhibitors was higher than 90%, as assessed by staining with trypan blue. To examine the signaling pathways involved in cytokine secretion, decidualized T-HESC cells were pretreated with 10µM SB203580 , 10 µM SP600125 , 2.5 µM BAY11-7082  or vehicle (DMSO). These reagents were added one hour before infection with Levels of human IL-6, IL-8, MCP-1, and TNF-α were measured in culture supernatants by sandwich ELISA, using paired cytokine-specific monoclonal antibodies according to the manufacturer’s instructions (BD Pharmingen).Each experiment was performed in duplicates on three independent occasions. The values obtained are presented as the mean ± SD. Statistical analysis was performed with one-way ANOVA, followed by Post Hoc Tukey’s Test or Dunnett’s Test using GraphPad Prism 6.0 software.


Moreover, the aerogels could retain 88% of their original shape after five cycles. The aerogel may be an appropriate material for the adsorption of heavy metal ions.Currently, the preparation of outstanding adsorbents has attracted public concern in environmentally friendly and sustainable pollutant redress. Herein, we report a directional freeze-drying method to prepare a strong and reusable adsorbent by introducing metal-organic framework which modified by ethylene diamine tetraacetic acid (named UiO-66-EDTA) into cellulose nanofiber (CNF) aerogel. Compared to traditional aerogels, the fabricated adsorbent showed a good flexibility and reusability by forming a homogeneous three-dimensional structure. By controlling the concentration of a crosslinkable carboxymethyl cellulose (CMC) solution, we produced aerogels with different pore structures and fibrillar, columnar, and lamellar morphologies. The obtained UiO-66-EDTA/CNF/CMC aerogel (U Sewage disposal is a global concern subject due to its destructive effect on ecologic sustainability. Wastewater, produced during industrial manufacturing for such goods as textiles, cosmetics, and chemical plants, contains many types of toxic dyes, oils, and heavy metal ions ,2,3,4. TAerogel, as a new generation of 3D network material, possesses many applications such as catalysis ,13,14, aMetal–organic frameworks (MOFs) are built from organic ligands and metal nodes, with tunable pore structures and large surface areas, and they have attracted much research attention in different application fields ,27,28,29-EDTACCA showed a high stability, as no obvious deformation was observed after several cycles. The prepared aerogels showed a great potential in practical applications for removing heavy metal ions.In this paper, directional freeze-casting was used to fabricate anisotropic CNF aerogels decorated with modified MOFs (UiO-66-EDTA). The obtained aerogels possessed different pore structures like fibrous, cylindric, or lamellar morphologies and showed both outstanding mechanical properties and a high removal efficiency. Moreover, the UN,N-dimethylformamide (DMF), and terephthalic acid  were achieved from Sinopharm Chemical Reagent Co. Ltd. Deionized water  were used to prepare aqueous solution.Bleached Douglas fir pulp was bought from Zhongshan NFC Bio-Materials Co., Ltd. , and 2,2,6,6-tetramethylpyperidine-1-oxyl , ethylenediamine tetraacetic acid disodium salt (EDTA-2Na), and sodium hypochlorite solution (NaClO) were bought from Alighting Reagent (Shanghai) Co., LTD. . Carboxymethyl cellulose , The synthesis process of CNF was taken from the literature . Firstly4 and 0.713 g of BDC were dissolved in 250 mL of DMF at room temperature, and the mixed solution was stirred vigorously. Simultaneously, acetic acid was dripped into the mixture to control the pH of the solution to faintly acidic. Then, the solution was kept at 120 °C for 24 h. The obtained powder was washed with water and methanol three times before being collected by centrifuge and being dried at 105 °C in the vacuum oven. To obtain the modified UiO-66, 0.1 g of UiO-66 was put into a 50 mL solution containing 3.8 wt% of EDTA. The mixed solution was kept at 60 °C for 24 h without any further operation so that the EDTA molecule could adsorb onto UiO-66. The final product UiO-66-EDTA was washed with DI water at 80 °C for 8 h and then further washed with methanol at 60 °C for 12 h. The UiO-66-EDTA was finally obtained after drying at 60 °C overnight.MOF (UiO-66) powder was prepared according to the literature . One gra-EDTACCA caused by small bubbles wrapped in the colloidal solution, the prepared mixed solution was put in a vacuum oven at 75 °C for 1 h before being transferred into freezing equipment. The final product was frozen using liquid nitrogen (−196 °C) via directional freeze equipment. The prepared samples were all cylinders with a bottom diameter and a height of 10 mm.First, 0.1 g of modified UiO-66-EDTA powder was added into a 10 mL CNF solution for sonication. Subsequently, CMC with different concentrations of 1, 2, and 4 wt% was added into the suspension (mass ratio of CNF:CMC = 1:1) and vigorously stirred for 1 h. To eliminate impact on the structure and the performance of the U−1. XRD was used to determine the crystalline structure of the hybrid aerogels using Cu Kα radiation as a source . The XPS of the samples was measured by using an AXIS UltraDLD  with Al Kα X-ray as the excitation source. The surface area of the MOFs and the modified MOFs were measured by the Brunauer–Emmett–Teller (BET) theory , and all the samples were pretreated at 105 °C for 10 h to degasify in a vacuum before testing.Inductively coupled plasma mass spectrometry  was used to measure the concentration of metal ions in the aqueous solution. FE-SEM was used to detect the morphology of the aerogels using a JSM-7600F field emission scanning electron microscope . The FT-IR of the samples were carried out with an FTIR Bruker VERTEX 80 spectrometer , and the spectral range of the IR experiment was 4000–600 cmP, %) of the U-EDTACCA aerogel was figured by Formulas (1) and (2):V represents the volume (cm3) of the aerogel, and The density  of the absorbents and the removal percentage E (%) were calculated using Equations (3) and (4), respectively:C0 (mg L−1) represents the initial concentration, tC (mg L−1) represents the concentration at time t, eC (mg L−1) represents the equilibrium concentrations, AM (g) represents the weight of adsorbent, and HV (L) represents the volume of heavy metal ion solutions.In this paper, CreQ (mg g−1) and tQ (mg g−1) represent the adsorption capacities at equilibrium and the adsorption capacities at time t (h), respectively, and k1 is the rate constant of the pseudo-first-order model. The pseudo-second-order kinetic model is used to fit the variation law of adsorption amount and time, wherein k2 is the rate constant of the pseudo-second-order kinetic model.The rate constant of adsorption corresponded Equations (5) and (6):-EDTACCA. The adsorbent was mainly formed by loading functional UiO-66-EDTA into a nanocellulose aerogel network. -EDTACCAs was formed (-EDTACCAs showed a low density (0.005 g cm−3), a high porosity (99%), and good mechanical properties, which greatly improves the application prospect of U-EDTACCAs in the field of heavy metal adsorption.s formed D. By con-EDTACCAs were examined by FE-SEM. -EDTACCAs. Apparently, there was a visible anisotropic pore structure not only on the vertical side but also on the horizontal side. In 2 adsorption/desorption isotherms and the BET surface area plots of UiO-66 and UiO-66-EDTA. All the samples were pre-treated at 102 °C for 10 h for degasification in a vacuum before testing. The BET surface areas of UiO-66 and UiO-66-EDTA were calculated to be 851 and 582 m2 g−1. In this paper, we found that the aerogel with a columnar structure performed well in adsorption, mechanical, and reuse, so the following performance tests were all columnar structure aerogels without special instructions.The surface morphology and alignment degree of U2+, Co2+, and Mn2+, respectively. To demonstrate that the crystalline form of UiO-66-EDTA was not destroyed by the modification, an XRD test was performed. A comparison of the diffraction peaks is shown in −1 was due to -OH stretching vibration. The peaks centered at about 1656–1395 cm−1 were due to the bond of the C=O stretching vibration in the carboxylic acid, the bond of C=C stretching vibration in a benzene ring, and the O–C–O stretching vibration in the BDC ligand. The peaks of 2901 and 1031 cm−1 were caused by the stretching vibration of C–H and C–O–C, respectively. The wide peak ranged from 1110 to 1164 cm−1 might have been caused by the asymmetric tensile vibration of the glycosidic ring. In addition, the FTIR spectra of UiO-66-EDTA showed that the newly appeared peak centered at 1193 cm−1 was caused by the C–N stretching band, which indicated that EDTA was successfully grafted onto UiO-66.XPS was used to examine the chemical components of the samples. Wide-scan XPS patterns of the samples are shown in -EDTACCA. U-EDTACCA was added into a 10 mL aqueous solution containing heavy metal ions with a concentration of 10 mg L−1 under the condition of pH = 5. As can be seen in -EDTACCA’s adsorption capacities (Qt) at different times. In this paper, the adsorption properties and reusability of U-EDTACCA were much better than those of pure CNF aerogels. The excellent adsorption ability could be attributed to the powerful chelating between target ions and EDTA in hybrid aerogels. -EDTACCA for each measured metal ion, and the results showed that the best removal rate of heavy metal ions was 98%. Considering the complex composition of wastewater, the U-EDTACCA was put into a mixed solution containing five kinds of metal ions to test its removal efficiency for mixed metal ions. The result is shown in -EDTACCA was a potential material for purifying wastewater containing various metal ions. For comparison, adsorbents prepared by similar materials are listed in -EDTACCA was the best. Above all, large pores at the micron level and the anisotropy pore structure of the CNF aerogels were beneficial for the fast removal of heavy metal ions and promoted the contact between functional UiO-66-EDTA particles and heavy metal ions. In addition, aerogels after adsorbing were easy to collect compared to pure MOFs in the powder form.In this paper, nine kinds of typical metal elements were selected to prepare the contaminated solution to evaluate the adsorption efficiency of U-EDTACCA aerogels were further measured via elemental mapping using SEM. In EDTACCA, which could be used as particular metal-connected sites for the command of metal ions’ dispersion in aerogel adsorbents. -EDTACCA. It was shown that Cr3+, Ni2+, Co2+, and Mn2+ were uniformly dispersed in the aerogels, thus providing good proof of our hypothesis.Heavy metal ion-loaded U-EDTACCA. Obviously, U-EDTACCA loaded with Cr3+ could recover its original shape after the mechanical squeezing process without any deformation, which demonstrated its excellent mechanical properties. It is noteworthy that the U-EDTACCA loaded with metal ions could be reused by washing it with a high concentration of the EDTA-2Na solution. 2+ ions, and it returned back to the white color after being washed with the EDTA-2Na solution. This phenomenon demonstrated that the prepared aerogels could be easily recycled without destroying their inner structure. The regenerated aerogels were further used as samples in the following metal ion removal experiments. The U-EDTACCA retained over 88% of its adsorption capacity for all the measured metal ions after five cycles  could maintain its original shape without damage.For practical application, the reusability of the samples was also tested. e cycles A. As sho-EDTACCA was prepared by combining the functional UiO-66-EDTA and CNF into an anisotropic aerogel followed by directional freeze-casting methods. The prepared aerogel exhibited a good absorptivity and outstanding shape stability under extrusion. By controlling the concentration of the CMC solution, aerogels produced three types of pore structures with fibrillar, columnar, or lamellar morphologies. The adsorption experiment for total nine kinds of heavy metal ions showed that the removal efficiency was more than 91%. Apparently, anisotropic pore structure was beneficial for improving the adsorption efficiency. Moreover, the U-EDTACCA showed excellent stability after five cycles without obvious deformation, and the removal efficiency was still over 88%. The U-EDTACCA, with good reusability and high adsorption properties, may have potential applications in the field of removing heavy metals.The novel adsorbent U


The use of lateral flow immunoassays (LFIAs) for rapid on-site testing is restricted by their relatively high limit of detection (LoD). One possible way to decrease the LoD is to optimize nanoparticle properties that are used as labels. We compare two types of Au nanoparticles: usual quasispherical gold nanoparticles (C-GNPs), obtained by the Turkevich–Frens method, and superspherical gold nanoparticles (S-GNPs), obtained by a progressive overgrowth technique. Average diameters were 18.6–47.5 nm for C-GNPs and 20.2–90.4 nm for S-GNPs. Cardiomarker troponin I was considered as the target analyte. Adsorption and covalent conjugation with antibodies were tested for both GNP types. For C-GNPs, the minimal LoD was obtained with 33.7 nm nanoparticles, reaching 12.7 ng/mL for covalent immobilization and 9.9 ng/mL for adsorption. The average diameter of S-GNPs varied from 20.2 to 64.5 nm, which resulted in a decrease in LoD for an LFIA of troponin I from 3.4 to 1.2 ng/mL for covalent immobilization and from 2.9 to 2.0 ng/mL for adsorption. Thus, we obtained an 8-fold decrease in LoD (9.9 to 1.2 ng/mL) by using S-GNPs. This effect can be related to more effective antibody immobilization and improved S-GNP optical properties. The obtained results can improve LFIAs for various practically significant analytes. Lateral flow immunoassay (LFIA)—also known as immunochromatography—has been suggested as an effective analytical method for point-of-care diagnostics ,2. The dVarious approaches have been considered for increasing LFIA sensitivity, including multistep analysis and specific detection techniques ,6,7. HowThe most widely used GNP synthesis method is based on sodium citrate’s reduction in gold salts. Turkevich  suggesteDespite the available wide range of GNP sizes, the question of the optimal size for LFIA is still under debate, including the impact of GNPs’ shape on this choice. Basically, the large sizes of GNPs allow a target (controlled) molecule to be labeled with a large number of gold atoms. However, the literature does not give a reason to believe that the differences in detection limits of immunochromatography are determined primarily and exclusively by GNP size. The comparative consideration of GNPs with different diameters in immunochromatography ,32,33,34Some experimental results show that 30–40 nm GNPs are optimal ,35,36 beFrom this point of view, superspherical GNPs (S-GNPs) can be more suitable labels for LFIA instead of the usual quasispherical nanoparticles obtained with the Turkevich–Frens method. The particles used for LFIA require, at least, high stability of colloidal dispersions, excluding their aggregation and nonspecific binding on the membrane. In this regard, the absence of fluctuations in the particle surface is an important potential advantage of superspherical GNPs. The unified surface properties of superspherical GNPs reduce their nonspecific interactions. Monodispersed colloids of S-GNPs can be obtained using seed-mediated growth in a cetyltrimetylammonium bromide solution ,44. The Here, we compare the LoD of LFIA strips based on S-GNPs and conventional C-GNP synthesized according to the Turkevich–Frens method. For a grounded comparison, a set of GNPs with different diameters was synthesized and conjugated with antibodies by using simple physical adsorption and covalent binding using a succinimide-thiol crosslinker. As an antigen, troponin I (cardiac isoform) was chosen, which is widely used in medical diagnostics as a biomarker of acute myocardial infarction . Due to 4, 99%), 3,3′,5,5′-tetramethylbenzidine dihydrochloride (TMB), tris(hydroxymethyl)aminomethane (Tris), Tween 20, Triton X-100, dimethyl sulfoxide (DMSO), sodium citrate, and sodium azide were purchased from Sigma–Aldrich . Tetrachloroauric acid  was procured from Alfa Aesar . Bovine serum albumin (BSA) came from Biowest . Thiolated polyethylene glycol  and orthopyridyl disulfide polyethylene glycol succinimidyl ester  were obtained from Creative PEG Works . All auxiliary reagents  were of analytical or chemical purity. All chemicals were used without further purification.The following antigens and antibodies were used in this study: native human troponin I , monoclonal anti-cTnI antibodies, IC4 and IC19 clones , and goat anti-mice immunoglobulin antibodies . Cetyltrimethylammonium bromide , cetyltrimethylammonium chloride , L-ascorbic acid , sucrose, sodium borohydride . To manufacture lateral flow test strips, the following membranes were used: a nitrocellulose (NC) membrane grade CNPC with a pore size of 15 μm attached to a solid support, conjugate release matrix PT-R7, sample membrane GFB-R4, and absorption membrane AP045 . ELISA was performed in transparent 96-well Costar 9018 polystyrene microplates .4 was added to deionized water, as indicated in Citrate-capped gold nanoparticles were synthesized via the Frens method  with mod4 (10 mM) to a mixture containing 5 mL of aqueous CTAB (0.2 M) and 5 mL of HAuCl4 (1 mM). Then, 10-nm GNPs were prepared by adding 20 mL of 0.5 mM HAuCl4 to a mixture containing 20 mL of CTAC (0.1 M), 15 mL of AA (0.1 M), and 0.5 mL of seeds. After 30 min of the reaction, the resulting 10-nm GNPs were centrifuged at least thrice at 20,000 g for 60 min. Finally, 10-nm GNPs were resuspended in 10 mL of 0.1 M CTAC. Then, these 10 nm GNPs were overgrown to a designed size. To this end, 0.1 M CTAC, 10 mM AA and the 10-nm GNPs were mixed, as indicated in 4 was added by using a syringe pump at the injection rate 10 mL/h. Finally, the S-GNPs were centrifuged at 1000–15,000 g and resuspended in water to have an optical density of about 1.5. The obtained colloids were labeled as S-GNPs-1–S-GNPs-5.The superspherical gold nanoparticles were made by using a protocol described elsewhere ,33. In tThe C-GNP preparations were applied to 300-mesh grids  coated with formvar film. Images were acquired with a CX-100 electron microscope  at an accelerating voltage of 80 kV. Digitized images were analyzed with Image Tool software. TEM images of S-GNPs were recorded with a Libra 120 microscope  at the Simbioz Center for the Collective Use of Research Equipment in the Field of Physical–Chemical Biology and Nanobiotechnology, IBPPM RAS, Saratov.Dynamic light scattering was measured using a Malvern Zetasizer Nano . Statistical data processing was performed by Malvern Softwarever 7.11 . Diameter determination of particles was performed in the range from 0.3 nm to 10 μm.v/v) were prepared before each experiment. Scandium was used as the internal standard, eliminating the fluctuations coming from the measuring conditions. All samples were prepared in triplicate. Quantum software  was used for data collection and processing. In the calculations of GNP concentrations, the Au density was equal to 19.3 g/cm3 and the volume of one particle was 4/3·πr3, where r was half the sum of the half-mean major and half-mean minor axis lengths obtained by TEM.The Au concentration in the C-GNP and S-GNP solutions was obtained by inductively coupled plasma mass spectrometry (ICP-MS), as described in our paper . The ICPw/v). GNPs with immobilized antibodies were separated from unreacted antibodies by centrifugation for 15 min at the accelerations indicated in w/v). The preparations were stored at 4–6 °C.GNPs were functionalized with anti-cTnI monoclonal antibodies and clone IC4. GNP solutions (pH 9.0) were added to antibody solutions at the proportions indicated in Covalent immobilization of the IC4 antibodies on GNPs was carried out according to our previous paper . First, w/v) was used. For the test zone (TZ), 1.0 mg/mL solutions of anti-cTnI antibodies and clone IC19 in the same buffer were used; 2.0 μL of both of the above solutions were applied per 1 cm of the working nitrocellulose membrane width. Conjugates of C-GNPs or S-GNPs with anti-cTnI antibodies, clone IC4, were applied to glass fiber PT-R7 membranes at dilutions corresponding to optical density 5.0 at 520 nm (16.0 μL per 1 cm of membrane width). The membranes with deposited immunoreagents were air-dried at room temperature for at least 20 h. These two membranes as well as the membrane for sample separation and the final adsorbing membrane were combined to assemble multimembrane composites, from which 3.5-mm-wide test strips were obtained using an automatic guillotine Index Cutter-1 .Reagents were applied to membranes comprising the assay system with an IsoFlow automatic dispenser . To form the control zone (CZ), a 1.0 mg/mL solution of GAMI antibodies in PBS containing 0.25% BSA, 0.25% sucrose, and 0.1% sodium azide  for 1 min and then placed on a horizontal surface. The result was checked within 10 min after sample application. The assaying of each sample was carried out in triplicate.Digital images of the test strips were obtained with a Canon CanoScan 9000F scanner and analyzed with TotalLab software , as described in our previous paper . The depC-GNP and S-GNP series were synthesized with varied ratios of reactants see  to reachFor further characterization of the obtained preparations, GNP size and shape were analyzed using TEM. 2.The next stage of the study was the immobilization of antibodies on the surface of the obtained GNPs. The choice of antibody: GNP ratio for immobilization was based on the calculation of the necessary quantity of immunoglobulin molecules to reach a monolayer coverage of the GNPs’ surface. To choose the optimal conditions for LFIA, the obtained conjugates with adsorbed and cross-linked antibodies were used as labels, and the intensity of the test zone was measured as a function of the cTnI concentration. The obtained data are shown in Based on the obtained concentration dependencies, the LoD values were determined for all four series of the conjugated GNPs, namely adsorption and covalent conjugates of C–GNPs and S-GNPs. These values are summarized in As can be seen in the case of using C-GNPs as labels, the most effective binding of conjugates in the test zone was obtained for C-GNP3 particles with an average size of 33.7 nm. Note that the adsorption immobilization of antibodies was more effective than the covalent binding. In contrast, S-GNP covalent immobilization of antibodies gives a more intense signal than adsorption. The optimal S-GNP size was 64.5 nm. The chosen variants are marked in red in Based on this choice, we further measured the LoD for LFIA strips with optimal GNP sizes, namely for conjugates C-GNPs-3–IC4  and S-GNPs-4–IC4  for both immobilization regimes. The images of test-strips for the assays of various cTnI concentrations are shown in The best variants for both GNPs demonstrate a significant increase in sensitivity . The posThis difference is caused by a combination of two factors: higher intensity of coloration for the same cTnI content and lower signal variation for the S-GNPs-based assays, providing reliable distinguishing of positive and negative samples for low levels of coloration. According to the intensity of the test zone coloration, the s-GNPs are twice as bright as c-GNPs .The presented data differ with the earlier published  comparisThe freshly prepared S-GNPs and C-GNPs demonstrated good colloidal stability with reproducible adsorption spectra and the absence of visible precipitates, independent of their size. The DLS data indicated the occasional presence of a small (0.1–0.5%) quantity of aggregates with diameters in the range of 100 nm–1 mkm . These affects were not in strong accordance with GNP type and did not lead to further increased aggregation see . More prPhysical antibody mechanisms bound to a GNP surface for different preparations need additional investigation using sophisticated equipment to estimate the orientation of immobilized antibodies, their conformational rearrangement, storage of antigen-binding ability, kinetic binding parameters, and steric hindrance for interaction with globular antigen molecules. The existing concepts consider surface defects at the atomic level and changing curvature as factors influencing possible partial inactivation of immobilized antibodies, but this interconnection has yet to be grounded as a priority and universal factor. In any case, two reasons should be taken into consideration when evaluating identified S-GNP benefits. The first is minimal differences in the properties of the S-GNPs’ surface, providing the possibility to maximize the cases of efficient antibody binding under the chosen optimal reaction media. The second is the use of additional stabilizers in the course of the two-step S-GNP synthesis. Due to this, the effect of colloidal instability is moved for S-GNPs to higher diameters, as compared with C-GNPs, and the intensity of coloration for labeling individual analyte molecules increased in accordance with the growth in the label’s size. Note that both effects are analyte-independent, which determines their high potential for LFIAs of other compounds.The presented study gives an experimental estimation of LODs that integrates both basic properties of the conjugates  and specific properties connected with aggregation stability, steric availability of a particular antigen for binding to immobilized antibodies, and so on. The compositions of all these factors show the need for more experimental studies. The identified regularities are impossible for prognostic assessment of the reactivity of conjugates and LODs achieved with their help.The presented study demonstrated a significant improvement in lateral flow immunoassay sensitivity by using superspherical gold nanoparticles instead of the commonly used quasispherical citrate-capped gold nanoparticles via the Turkevich–Frens technique. The known modifications of C-GNPs synthesis do not give such monodispersity as the super-spherical preparation under consideration in this paper. The proposed superspherical GNPs have advantages in unified size and shape that are unattainable for alternative preparations. Therefore, these GNPs were compared with the common Turkevich–Frens C-GNPs. They caused a big gain in sensitivity in the immunochromatographic analysis.This improvement is the result of two factors: (i) more efficient immobilization of antibodies on the nanoparticle surface without variations in curvature and (ii) the possibility of using larger nanoparticles with increased quantity of binding sites of immobilized antibodies. The resulting lowering of the detection limit for troponin I gives reason for further extension of the field of application to other practically important analytes.


Child growth impairments are rampant in sub-Saharan Africa. To combat this important health problem, long-term follow-up studies are needed to examine possible benefits and sustainability of various interventions designed to correct inadequate child growth. Our aim was to perform a follow-up study of children aged 60−72 months whose mothers participated in a two-armed cluster-randomized education intervention trial lasting 6 months in rural Uganda when their children were 6−8 months old with data collection at 20−24 and at 36 months. The education focused on nutrition, hygiene, and child stimulation.We measured growth using anthropometry converted to z-scores according to WHO guidelines. We also included assessments of body composition using bioimpedance. We used multilevel mixed effect linear regression models with maximum likelihood method, unstructured variance-covariance structure, and the cluster as a random effect component to compare data from the intervention  with the control group .Of the 511 children included in the original trial, data from 166/263 (63%) and 141/248 (57%) of the children in the intervention and control group, respectively, were available for the current follow-up study. We found no significant differences in any anthropometrical z-score between the two study groups at child age of 60−72 months, except that children in the intervention group had lower (P = 0.006) weight-for-height z-score than the controls. There were no significant differences in the trajectories of z-scores or height growth velocity (cm/year) from baseline  to child age of 60−72 months. Neither did we detect any significant difference between the intervention and control group regarding body composition  at child age 60−72 months. Separate gender analyses had no significant impact on any of the growth or body composition findings.In this long-term study of children participating in a randomized maternal education trial, we found no significant impact of the intervention on anthropometrical z-scores, height growth velocity or body composition.Clinical Trials  ClinicalTrials.gov ID NCT 02098031. This anthropometric deficiency is termed stunting and is defined as height-for-age z-score (HAZ) more than two standard deviations below the median of the WHO child growth standard . A recenThe widespread use of stunting as an indicator of linear growth failure and as a predictor of negative health outcomes and mortality later in life, largely stems from its robustness and low cost for use in demanding resource-strained settings. However, stunting alone does not necessarily capture how adverse nutritional exposures affect more refined aspects of growth, impact on the dynamics in child growth over time and on subsequent risk of disease in adolescent and adult life . In addiPrimarily to prevent impaired linear growth, we conducted the “Child Nutrition and Development Study” (CHNUDEV) in 2013−14, a two-armed, pragmatic cluster-randomized controlled trial (RCT). In that trial we examined child anthropometrical effects of a maternal education intervention focusing on nutrition, hygiene and child stimulation in South-Western Uganda, a part of the country with high prevalence of stunting ,10. The Monitoring child growth patterns over time is essential to evaluate long-term effects of interventions given at early child ages. Thus, we have now performed a follow-up of our RCT cohort when the children reached 60−72 months, i.e. at the time of school-start. This unique longitudinal data set allowed description of both anthropometry and growth velocity trajectories. In addition, we collected body composition data at 60−72 months age.22.1The RCT was approved by the Uganda National Council for Science (HS 1809), the AIDS Support Organisation Research Ethics Committee (No. TASOREC/06/15-UG-REC-009), and the Norwegian Regional Committee for Medical and Health Research Ethics (no. 2013/1833).2.2The RCT was conducted between October 23, 2013 and February 16, 2014 in the neighbouring districts of Kabale and Kisoro in South-Western Uganda because of high stunting rates . Briefly2.3An education intervention emphasizing nutrition, hygiene  and stimulation was delivered to mothers in the intervention group as described in the 2.4Height, weight, and mid-upper arm circumference at each follow-up sampling time were measured according to WHO guidelines by trained nutritionists as detailed previously ,11. WeigHeight growth velocity was calculated as the difference between the height values (cm) at each follow-up age  and the baseline height values divided by the follow-up time in years.2.5Body composition at 60−72 months age was estimated using a dual frequency (6.25 and 50 kHz) bioimpedance analyzer . The measurements were conducted in the morning among non-fasting children with light clothing.2.6Whereas the RCT recruited in total 511 mother/child pairs, the minimum calculated number of such pairs was 352 to achieve the primary outcome, i.e. to detect a difference of 0.3 SD  in HAZ at 20−24 months of age between the intervention and control group as described in the The anthropometric z-scores at baseline (when the RCT started with the children aged 6−8 months) and when the children reached 60−72 months of age, were calculated using the 2006 and 2007 WHO growth references, respectively. We computed the following z-scores as  for height-for-age (HAZ), weight-for-age (WAZ), weight-for-height (WHZ) and mid-upper arm circumference-for-age (MUACZ). Since the 2007 WHO growth reference does not have MUACZ for age references, we used the method of Mramba et al. to calculate MUACZ at 60−72 months of age . UnderweThe analyses used the intention-to-treat approach and all the statistical tests were two-sided. We calculated anthropometric and body composition means  for each study group (intervention and control) at 60−72 months of age and their cluster-adjusted mean differences. We used multilevel mixed effect linear regression models with maximum likelihood method, unstructured variance-covariance structure and the cluster as a random effect component to compare the intervention with the control group for all the continuous anthropometric and body composition measurements at 60−72 months of age. To compare nutritional status (i.e. using anthropometry as a proxy) grouped as binary (using the respective z-scores < −2) between the intervention and control group, multilevel mixed effect logistic regression models with cluster as a random effect component were used.In addition, we compared the mean change (gain/loss) of the anthropometric measurements in each of the randomization group from baseline (6−8 months) to 60−72 months age accounting for clustering and also adjusted for the regression-to-the mean. We calculated the regression-to-the mean as the difference between baseline individual anthropometric measurements values and their group baseline (6−8 months) mean . Statist33.1For this follow-up of the children aged 60−72 months, 166 (mean (SD) age 71.4 (1.9) months) and 141 (mean (SD) age 70.9 (1.7) months) from the intervention and control group (P = 0.47) of the total RCT cohort (n = 511) could be included, respectively . Among t3.2Since we lacked data from about 40% of the 511 children enrolled into the RCT, we first examined the differences in baseline anthropometrical values between the control and intervention group of the follow-up cohort . ImportaNext, we found no significant differences in mean HAZ, WAZ or MUACZ at child age 60−72 months between the control and intervention group . In contWe then examined the mean changes in anthropometrical z-scores from baseline to 60−72 months stratified by study group affiliation. The changes in the four anthropometrical z-scores  were all approximately at or below zero, indicating no average catch-up growth over these 54 months . Of noti3.3The absolute height growth velocity was not significantly different between the control and intervention group at any of the three sampling time points during the follow-up period . As expe3.4We then evaluated the anthropometrical z-scores  as markers of nutritional status. At child age 60−72 months, approximately one-third; i.e. 49 (30%) and 43 (31%) (P = 0.86) among children randomized to the intervention and control group, respectively, were stunted. At 60−72 months, wasting was rare; only 1 (0.7%) child from the control group was wasted and none in the intervention group. In contrast, at 60−72 months, more children in the intervention compared with the control group were classified as underweight: 28 (17%) vs. 9 (6.4%); yielding a cluster-adjusted odds ratio of 2.96 .3.5To complement our anthropometrical data, we also included measurements of body composition using bioimpedance. 4Here we present data of a long-term (~6 years) follow-up of a randomized maternal education trial primarily designed to prevent impaired linear growth among small children in rural Uganda. Similar to our previous follow-up studies of this trial cohort, we could not detect any significant effect of the intervention on child anthropometrical z-scores  ,12. In tNumerous studies have addressed one or several drivers of impaired growth, but usually with modest or no improvement . AlthougWhereas there has been some progress in preventing or reducing impaired linear growth (low HAZ) in many low- and middle-income countries, stunting is still rampant in sub-Saharan Africa ,5,21,22.Accumulating evidence suggests that undernutrition and over-weight/obesity share characteristics in terms of trends and underlying determinants ,28. In lWe detected a significantly higher fraction of children that were underweight in the intervention group compared with the controls at 60−72 months, but there were no significant changes in the other anthropometric measures or in body composition, and the 95% confidence interval for the estimated odds ratio was also quite wide.Interestingly, the concept of stunting as a marker for undernutrition has recently been questioned by Scheffler et al. ,31. TheyMuch focus is on preventing rapid gain of fat mass in infancy to decrease the risk of non-communicable disorders such as cardiovascular disease, diabetes type 2 and the metabolic syndrome, as recently reviewed ,38. We tThe major strengths of this study are the robust and pragmatic design of the original RCT and the long-follow-up period. The adherence to the intervention was probably adequate as mothers in the intervention group demonstrated more relevant knowledge after the intervention period, compared with the controls . AdditioIn conclusion, in this six-year follow-up study of children participating in a randomized maternal education trial, we found no significant impact of the intervention on anthropometrical z-scores, height growth velocity or body composition. This trial cohort should be re-examined when entering into adulthood (i.e. at about 18 years of age), and preferably be supplemented with assessments of various growth biomarkers.Conceptualization, P.A, G.M, A.C.W. and P.O.I.; Data curation, P.A., X.C. and G.M.; Formal analysis, G.M. and M.N.; Methodology, P.A, M.N., G.M, A.C.W. and P.O.I.; Project administration, P.A., P.O.I. and G.M.; Resources, P.O.I.; Writing-original draft, M.N., A.C.W. and P.O.I.; Writing-review & editing, P.A., M.N., X.C., A.C.W., P.O.I. and G.M.CONSORTFigure S1Figure S2Supplementary methodsTable S1Table S2Table S3


It has been claimed that efficacy estimates based on the Hamilton Depression Rating-Scale (HDRS) underestimate antidepressants true treatment effects due to the instrument’s poor psychometric properties. The aim of this study is to compare efficacy estimates based on the HDRS with the gold standard procedure, the Montgomery-Asberg Depression Rating-Scale (MADRS).We conducted a meta-analysis based on the comprehensive dataset of acute antidepressant trials provided by Cipriani et al. We included all placebo-controlled trials that reported continuous outcomes based on either the HDRS 17-item version or the MADRS. We computed standardised mean difference effect size estimates and raw score drug-placebo differences to evaluate thresholds for clinician-rated minimal improvements . We selected 109 trials  that assessed the HDRS-17 and 28 trials  that assessed the MADRS. The summary estimate (effect size) for the HDRS-17 was 0.27 (0.23 to 0.30) compared to 0.30 (0.22 to 0.38) for the MADRS. The effect size difference between HDRS-17 and MADRS was thus only 0.03 and not statistically significant according to both subgroup analysis (p = 0.47) and meta-regression (p = 0.44). Drug-placebo raw score difference was 2.07 (1.76 to 2.37) points on the HDRS-17  and 2.99 (2.24 to 3.74) points on the MADRS .Overall there was no meaningful difference between the HDRS-17 and the MADRS. These findings suggest that previous meta-analyses that were mostly based on the HDRS did not underestimate the drugs’ true treatment effect as assessed with MADRS, the preferred outcome rating scale. Moreover, the drug-placebo differences in raw scores suggest that treatment effects are indeed marginally small and with questionable importance for the average patient. The debate whether antidepressants are an effective treatment for depression is ongoing and unresolved –5. AlthoAn alternative approach to examine the efficacy of antidepressants would be to base effect size estimates on an outcome that is widely accepted as a reliable and valid measure of depression. One such outcome is the Montgomery-Asberg Depression-Rating Scale (MADRS), which was constructed to be particularly sensitive to change and to treatment effects on core depression symptoms . IndepenThe aim of this meta-analysis was thus to re-evaluate the data from short-term antidepressant trials for adults with major depression collected by Cipriani et al.  by focusSince this a post-hoc analysis of a freely available dataset, we did not write a study protocol and did not pre-register the planned analysis. That is, we did not conduct our own literature search, but relied on the work by Cipriani et al. . Except https://data.mendeley.com/datasets/83rthbp8ys/2). This is the largest database of new-generation antidepressants for the acute treatment of major depression compiled thus far. The comprehensive literature search conducted by Cipriani et al. [Our analysis was based on short-term, mostly 8-week clinical trials of antidepressants for adults with unipolar major depression, reported in a recent systematic review by Cipriani et al. . The auti et al.  yielded When trials had multiple treatment arms with different doses of antidepressants, these arms were aggregated. This is an appropriate procedure, as low-to-medium doses achieve the highest efficacy . When trWe calculated the standardised mean differences for each trial and aggregated them by means of a random effect meta-analysis based on the inverse variance method according to the procedure suggested by Munkholm et al.  using th2. Test statistics and confidence intervals were adjusted according to the procedure by Hartung and Knapp, as recommended in the Cochrane handbook [We used subgroup analysis and meta-regression to test for differences between the HDRS and MADRS. Effect size estimates with corresponding 95% confidence intervals (CI) were based on Hedges’ g standardised mean difference method and the variability of effect sizes due to heterogeneity was calculated as Ihandbook .Classifying effect sizes as small or large based on arbitrary thresholds is not recommended . We therA sensitivity analysis included a meta-regression across all antidepressants and controlled for potential confounders that differed between trials using the HDRS-17 and the MADRS. These covariates included year of publication, drop-out-rate, sample size, and publication status. We also cross-validated our findings with the standard methods for continuous outcomes proposed in the Cochrane handbook, the DerSimonian-Laird random-effects meta-analysis and the inverse-variance fixed-effects method .For the overall analysis across different antidepressants, there were 137 trials/comparisons. A full list of included studies is provided in the online supplement  and for 2 = 45%) and moderate for the MADRS (I2 = 74%). The forest plot for the meta-analysis across all drugs is shown in the online supplement  for the HDRS-17 and the MADRS were almost identical , and the difference was statistically not significant based on both subgroup analysis  and meta-regression . Observed variability across studies, which could indicate between-study heterogeneity, was low for the HAMD-17  was possible for bupropion (k = 9), duloxetine (k = 17), escitalopram (k = 12), paroxetine (k = 21), venlafaxine (k = 7), and vilazodone (k = 7). The results for the individual drugs are shown in 2: heterogeneity index, p: p-value of the meta-regression analysis with the instrument (HDRS-17 vs. MADRS) as predictor variable, k: number of trials, N: number of participants (antidepressant plus placebo arm).SMD (95% CI): standardized mean difference based on Hedges’ g and corresponding 95% confidence interval, IAs detailed in the methods section, the pre-defined threshold for a minimal improvement according to clinician evaluation is 7 points on the HDRS and 8 points on the MADRS. Corresponding cut-offs for a minimal important difference according to patient ratings are about 4 points for the HDRS-17 and 5 points for the MADRS. In the present meta-analysis, the drug-placebo difference in raw scores was 2.07 (CI = 1.76–2.37) for the HDRS-17 (possible range 0–52 points) and 2.99 (CI = 2.24–3.74) for the MADRS (possible range 0–60 points) . Drug-plIn univariate meta-regression analysis, trials using the HDRS-17 and the MADRS differed by year of publication, dropout rate, and sample size, but not by publication status. In a multivariable meta-regression analysis that included all significant confounding variables as additional predictor variables, the overall effect of the instrument (HDRS-17 compared to MADRS) was statistically not significant and very small . The results were almost identical when different meta-analytic methods were used. The inverse-variance fixed-effects model produced estimates of ES = 0.25 (0.23–0.28) and ES = 0.30 (0.27–0.34) for HDRS-17 and MADRS, whereas based on the DerSimonian-Laird random-effects model the estimates were ES = 0.26 (0.23–0.29) and ES = 0.30 (0.22–0.38).Our meta-analysis found a mean effect size of 0.30 (0.22–0.38) for the MADRS as compared to 0.27 0.23–0.30) for the HAMD-17. That is, the effect sizes for both rating scales are comparable when averaged over all drugs. Although the MADRS is considered the gold standard  and the –0.30 forWe cannot preclude with certainty that there is no difference between HDRS-17 and MADRS for all individual drugs. After correction for multiple testing, for vilazodone the effect size based on MADRS was statistically significantly larger than the effect size produced by the HDRS-17. However, it is important to note that vilazodone’s HDRS-17 effect size (ES = 0.09) was considerably lower than the summary estimate for all drugs (ES = 0.27), whereas vilazodone’s MADRS effect size (ES = 0.41) was above the average for all drugs (ES = 0.30). This is a peculiar inconsistency, because if vilazodone was truly more efficacious than other drugs, this should show in an above-average score for both HDRS-17 and MADRS, but not in a very low HDRS-17 score and in a high MADRS score. This inconsistency might be explained by the low proportion of published vilazodone trials using the HDRS-17 , whereas all three trials using the MADRS were published (100%). It is known that the trials published in the scientific literature exaggerate efficacy estimates , 29. ThePrevious research tried to minimise biases assumed to underlie the full HDRS scale by focusing on selected items. For instance, Bech developed a HDRS subscale that contains only core depression symptoms . In a reHieronymus et al.  chose anThis study is not without limitations. First, it is important to note that the MADRS was applied in relatively few trials and comparisons between MADRS and HDRS-17 for individual drugs are thus limited to bupropion, duloxetine, escitalopram, paroxetine, venlafaxine, and vilazodone. However, it is unlikely that this restriction has introduced systematic bias. Gartlehner et al.  concludeThe effect size estimates reported in recent meta-analyses of antidepressants that were mostly based on the highly criticised HDRS-17 do not appear to underestimate the drugs treatment effects. When efficacy estimates are based on the MADRS, the preferred rating scale for depression that is particularly sensitive to change in core depression symptoms, the effect size estimates do not differ meaningfully. Moreover, we found that the average drug-placebo difference in raw scores for both the HDRS-17 and MADRS fall short of the cut-offs for a minimally important improvement. Prominent claims that the HDRS has significantly underestimated antidepressants’ true efficacy are thus not based on convincing evidence. A more plausible explanation would be that the efficacy of antidepressants is indeed poor and of questionable importance to the average patient.S1 Checklist(DOC)Click here for additional data file.S1 File(PDF)Click here for additional data file.